The present invention lies in the field of anti-cancer (gene) therapy. In particular, the invention relates to selective killing of (solid) tumor cells in a mammal by gene delivery via the blood circulation.
Many different kinds of solid tumors occur in the body of mammals, including humans. In many cases these tumors are extremely difficult to treat, especially in advanced cancer with metastases. Currently available therapies include surgery, radiation therapy, chemotherapy, radio-immunotherapy, cytokine treatment and hyperthermia. All these therapies have important limitations and disadvantages. E.g., surgery can only be performed on localized, accessible tumors; radiation and chemotherapy are associated with both acute and latent toxicity, and responses are often limited; radio-immunotherapy and hyperthermia have limited application and effectivity; and cytokine administration is often associated with toxicity and evokes many pleiotropic side-effects. Often said therapies are combined to improve efficacy and to decrease toxic side-effects. However, in general, the effectivity of said therapies and their combinations is still unsatisfactory.
More recently, gene therapy has been proposed as a novel approach to treat malignancies. The concept of gene therapy comprises the introduction of a molecule carrying genetic information into cells of a host, whereby said genetic information has a functional format. Said genetic information may comprise a nucleic acid molecule that encodes a protein. In this case said functional format means that the protein can be expressed by the machinery of the host cell. The genetic information may also comprise or encode nucleic acid molecules with a sequence that is complementary to that of a nucleic acid molecule present in the host cell. The functional format in this case is that the introduced nucleic acid molecule or copies made thereof in situ are capable of base pairing with the complementary nucleic acid molecule present in the host cell. Said genetic information may furthermore comprise a nucleic acid molecule that encodes or is itself a so-called ribozyme or deoxyribozyme. In this case said functional format means that said nucleic acid molecule or copies made thereof in situ are capable of specifically cleaving a nucleic acid molecule present in the host cell. Said genetic information may furthermore comprise a nucleic acid molecule that encodes or is itself a so-called decoy molecule. In this case said functional format means that said nucleic acid molecule or copies made thereof in situ (nucleic acid molecules or proteins) are capable of specifically binding a peptide molecule present in the host cell.
Said introduction of a molecule carrying genetic information into cells of a host is achieved by various methods known in the art. Said methods include, but are not limited to, direct injection of naked DNA constructs, bombardment with gold particles loaded with said constructs, and macromolecule mediated gene transfer using, e.g., liposomes, biopolymers, and the like. Preferred methods use gene delivery vehicles derived from viruses, including but not limited to adenoviruses, retroviruses, vaccinia viruses and adeno associated viruses. Because of the much higher efficiency as compared to e.g. vectors derived from retroviruses, vectors derived from adenoviruses (so-called adenoviral vectors) are the preferred gene delivery vehicles for transferring nucleic acid molecules into host cells in vivo.
The adenovirus genome is a linear double-stranded DNA molecule of approximately 36000 base pairs. The adenovirus DNA contains identical Inverted Terminal Repeats (ITR) of approximately 100 base pairs with the exact length depending on the serotype. The viral origins of replication are within the ITRs exactly at the genome ends. Adenoviruses can be rendered replication defective by deletion of the early-region 1 (E1) of their genome. Vectors derived from human adenoviruses (so-called adenoviral vectors), in which at least the E1 region has been deleted and replaced by a gene-of-interest, have been used extensively for gene therapy experiments in both pre-clinical and clinical phase. Apart from replication defective adenoviral vectors, helper independent or replication competent vectors, either or not containing a gene-of-interest, can also be used for gene therapy purposes. Adenoviral vectors have a number of features that make them particularly useful for gene therapy for malignancies. These features include (1) the biology of adenoviruses is characterized in detail, (2) adenoviruses are not associated with severe human pathology, (3) adenoviruses are extremely efficient in introducing their DNA into host cells, (4) adenoviruses can infect a wide variety of cells and have a broad host-range, (5) adenoviral vectors allow insertion of relatively large fragments of foreign DNA, (6) adenoviruses can be produced in large quantities with relative ease, and (7) adenoviral vectors are capable of transferring nucleic acid molecules very efficiently into host cells in vivo (Brody and Crystal, Ann. N. Y. Acad. Sci. 716(1994):90-101).
The present inventors and their coworkers as well as others have demonstrated that recombinant adenoviral vectors efficiently transfer nucleic acid molecules to the liver of rats (Herz and Gerard, Proc. Natl. Acad. Sci. U.S.A., 96 (1993):2812-2816) and to airway epithelium of rhesus monkeys (Bout et al., Gene Ther., 1 (1994):385-394; Bout et al., Hum. Gene Ther., 5(1994):3-10). In addition, the present inventors, their coworkers and others have observed a very efficient in vivo adenoviral vector mediated gene transfer into a variety of established solid tumors in animal models (lung tumors, glioma) and into human solid tumor xenografts in immune-deficient mice (lung) (Haddada et al., Biochem. Biophys. Res. Comm. 195 (1993):1174-1183; Vincent et al., Hum. Gene Ther., 7 (1996):197-205; reviewed by Blaese et al., Cancer Gene Ther., 2 (1995):291-297. Thus, preferred methods for in vivo gene transfer into tumor cells of nucleic acid molecules that encode molecules that can be used to kill said tumor cells make use of adenoviral vectors as gene delivery vehicles.
Said molecules that can be used to kill tumor cells include but are not restricted to suicide enzymes that convert a non-toxic prodrug into a toxic compound (e.g. the HSV-tk/ganciclovir system), cytokines, antisense nucleic acid molecules, ribozymes, and tumor suppressor proteins. In addition, treatment of cancer by gene therapy methods also includes the delivery of replicating vectors that are toxic to the tumor cells by themselves.
Gene therapy by introduction of nucleic acid molecules encoding suicide enzymes has been widely tested on a variety of tumor models. Especially the transfer of the Herpes simplex virus thymidine kinase (HSV-tk) gene into tumor cells in conjunction with systemic administration of the non-toxic substrate ganciclovir has proven to be an effective way of killing tumor cells in vivo (Esandi et al., Gene Ther., 4 (1997) :280-287; Vincent et al., J. Neurosurg., 85 (1996) :648-654; Vincent et al., Hum. Gene Ther., 7 (1996) :197-205). An important advantage of the HSV-tk/ganciclovir system is that upon ganciclovir treatment HSV-tk transduced tumor cells mediate a significant killing effect on neighboring untransduced tumor cells, the so-called bystander effect (Culver et al, Science 256 (1992) :1550-1552). Thus, using this approach there is no absolute need for gene transfer into every individual cell in a solid tumor to achieve successful gene therapy. A limitation of this approach, however, is that the effect remains local. Consequently, the HSV-tk gene needs to be delivered into every individual solid tumor or metastasis throughout the body.
Gene therapy for cancer by the introduction of nucleic acid molecules encoding cytokines is based on the concept of enhancing the immune response against the tumor cells. The ultimate goal of this approach is to obtain regression of the treated tumor and simultaneously induce such a high degree of immunity that coexisting metastases are also destroyed. The mechanism by which the cytokine enhances the immune response against the tumor cells most likely in many cases involves eliciting an inflammatory type cell infiltration that results in improved antigen presentation. During the local inflammation invading cells may lyse the tumor cells, releasing tumor antigens in a form that can be presented by other subpopulations of the invaders to T lymphocytes. These, in their turn could act against coexisting metastases. Compared to administration of a cytokine protein the gene transfer approach has the important advantage of high-level production of the cytokine at the site of the tumor, while systemic concentrations of the cytokine remain low. This avoids any pleiotropic and toxic side effects associated with said cytokine. Signs of (partially) successful cancer treatment have been obtained with tumor cells expressing IL-2 (Fearon et al., Cell 60: 397-401, 1990; Gansbacher et al., J. Exp. Med. 172:1217, 1990), IL-4 (Golumbek et al., Science 254:713-716, 1991; Platzer et al., Eur. J. Immunol. 22:1729-1733, 1992), interferon-gamma (Gansbacher et al., Cancer Res. 50:7820-7824, 1990), interferon-alpha (Ferrantini et al., Cancer Res. 53:1107-1112, 1993), TNF alpha (Blankenstein et al., J. Exp. Med. 173:1047-1052, 1991), IL-7 (Hock et al., J. Exp. Med. 174:1291-1298, 1991; McBride et al., Cancer Res. 52:3931-3937, 1992), G-CSF (Colombo et al., J. Exp. Med. 173:889-897, 1991), GM-CSF (Dranoff et al., Proc. Natl. Acad. Sci. U.S.A. 90:3539-3543, 1993), IL-12 (Tahara et al., Cancer Res. 54:182-189, 1994), IL-1 (Apte et al., In: Cytokine-induced tumor immunogenicity, Acad. Press, London, pp. 97-112, 1994; Apte et al., Folia Biol. Praha 40:1-18, 1994; Douvdevani et al., Int. J. Cancer 51:822-830, 1992; Nakata et al., Cancer Res. 48:584-588, 1988; Zxc3x6ller et al., Int. J. Cancer 50:443-449, 1992) and IL-3 (McBride et al., Folia Biol. Praha 40:62-73, 1993; Pulaski et al., Cancer Res. 53:2112-2117, 1993). The present inventors and their coworkers have previously observed partial regression of a non-immunogenic solid tumor (L42 non small cell lung cancer; Kal et al., NCI Monographs 6:111-114, 1988; Kal et al., Radiother. Oncol. 6:231-238, 1986; Kal et al., J. Natl. Cancer Inst. 76:943-946, 1986) growing subcutaneously in WAG/Rij rats after intra-tumor injection of adenoviral vectors expressing IL-1a or IL-3. This regression occurred both in the injected tumor and in an untreated distant (contralateral) L42 tumor (patent application EP 96.202725, incorporated herein by reference).
Interleukin-3 (IL-3) is a cytokine well described as a hematopoietic growth factor that has a wide range of target cells including progenitor cells of every lineage, excluding cells committed to the T and B lymphoid lineage (Schrader et al., In: Lymphokines, Acad. Press, San Diego, 1988). The main production of IL-3 by activated T cells has led to the hypothesis that IL-3 is not involved in steady-state hematopoiesis but functions as a link between on one hand the T lymphocytes of the immune system, which sense invasion of the body by foreign materials, and on the other hand the hematopoietic system which generates the cellular elements that mediate defense and repair responses (Ihle, In: Immunoregulatory cytokines and cell growth, Karger, Basel, 1989). IL-3 exerts a broad spectrum of biological properties (Ihle et al ., J. Immunol. 131:282, 1983), including stimulatory activity on several myeloid leukemia cell lines, formation of granulocyte-macrophage colonies, mast cell growth factor activity, P cell-stimulating activity and histamine producing cell-stimulating factor activity. In addition, IL-3 is capable of promoting the proliferation of megakaryocyte colony-forming cells, of supporting the differentiation of eosinophils and pre-B-cell precursors, of supporting proliferation of Natural Cytotoxic (NC) cells but not Natural Killer (NK) cells and of promoting the formation of osteoclasts. IL-3 also stimulates the effector functions of monocytes, eosinophils and basophils, thereby having the potential to regulate inflammation and allergy (Elliott et al., J. Immunol. 145:167, 1990; Haak-Frendesco et al., J. Clin. Invest. 82:17, 1988; Lopez et al., J. Cell. Physiol. 145:69, 1990). Human endothelial cells express the IL-3 receptor which expression is enhanced by tumor necrosis factor alpha (TNF-xcex1). IL-3 stimulation of TNF-xcex1-activated endothelial cells enhances IL-8 production, E-selectin expression and neutrophil transmigration (Korpelainen et al., Proc. Natl. Acad. Sci. U.S.A. 90:11137, 1993). This suggests that IL-3 plays a role in inflammation not only by stimulating effector functions of mature leukocytes but also by regulating their localization to sites of inflammation through its action on the endothelium.
There are several ways to administer recombinant adenoviral vectors with therapeutic genes into solid tumors that grow in a mammalian animal body. Currently, cancer gene therapy protocols predominantly use direct injection of the recombinant vector into the tumor (e.g., Haddada et al., Biochem. Biophys. Res. Comm. 195:1174-1183, 1993; Vincent et al., Hum. Gene Ther. 7:197-205, 1996). The major disadavantage of this application route is that metastases, and in particular micrometastases, in advanced cancer are practically impossible to reach with this approach. Therefore, such a gene therapy relies solely on a distant (immune mediated) effect of the introduced genetic information. Using current technology, said distant effect may not be expected to be complete and, consequently, may not be expected to cure the disease.
An alternative, and possibly better way of delivering genetic material into solid tumors and/or their metastases could be by administering the recombinant adenovector via the blood or lymphatic circulation. All established tumors, both primary and metastatized, that are larger than a few millimeter in diameter are vascularized (Folkman et al., J. Nat. Cancer Inst. 82:4, 1990; Folkman and Shing, J. Biol. Chem. 267:10931-10934, 1992). In addition, distant metastases usually emerge after migration of tumor cells from the primary tumor through the blood or lymphatic circulation. Thus, all solid tumors are in close contact with the circulation and, in priciple, could be reached via the circulation. Moreover, killing of a solid tumor does not neccessarily depend on gene transfer into the tumor cells themselves. Gene therapy strategies have been proposed where genetic material (e.g., the HSV-tk gene) is introduced into endothelial cells of the tumor vasculature (e.g., WO96/21416). This should result in destruction of the tumor vasculature, ultimately leading to tumor necrosis.
The total capillary surface area in an adult human is approximately 100 m2 comprising approximately 1012 endothelial cells, whereas the endothelial cell content of the vasculature of a solid tumor is about 4-log less (Chan and Harris, In: The Internet book of Gene Therapy. Cancer Therapeutics, eds. R. E. Sobol and K. J. Scanlon, 1995, Appleton and Lange, CT, pp. 211-227). Based on these estimations, intravascularly administered adenoviral vectors only have a 0.01% chance of interacting with endothelial cells in the vasculature of a distant tumor. The proliferation index of endothelial cells in the vasculature of a tumor is about 100-fold higher than that of normal endothelial cells (Hobson and Denekamp, Br. J. Cancer, 1984, 49:405-413). Thus, if gene delivery would preferentially occur into actively proliferating cells, the gene transfer efficiency into the chosen target cells could be raised to approximately 1%. However, because adenoviral vectors, in contrast to retroviral vectors, transduce both replicating and non-replicating cells, the estimate of 0.01% gene transfer into cells of the tumor vasculature is more realistic. In any event, administering the adenovector via the circulation is expected to result in at least 99% of the adenoviral vectors interacting and possibly being taken up by cells in normal tissues. This is highly undesirable with respect to toxic side-effects of the procedure. E.g., the introduction and expression of a suicide gene or an inflammation eliciting cytokine gene should obviously not take place in the endothelium of the normal vasculature. Therefore, until the present invention the common belief in the field has been that administering the adenovector to the tumor via the circulation requires some sort of specific targeting of the adenovector to the tumor or its vasculature (e.g., WO 96/25947). Said specific targeting may include specific interaction with and uptake by the intended target cells, as well as specific expression of the introduced genetic information in the intended target cells. Said specific targeting was felt to be necessary to ensure efficient gene transfer and to avoid toxic side-effects in other tissues. Many different molecules that are specifically expressed or upregulated on the cell surface of tumor cells or their vascular endothelial cells have been proposed as targets for specific uptake of gene transfer vectors. Examples of such molecules are carcinoembryonic antigen (CEA; Walther et al., Head-Neck 15:230-235, 1993), surface-bound vascular endothelial growth factor (VEGF; Plate et al., Int. J, Cancer 59(1994):520-529; Brown et al., Hum. Pathol. 26(1995):86-91), the avb3 integrin (Brooks et al., Science 264(1994):569-571), endosialin (Rettig et al., Proc. Natl. Acad.Sci. USA 89(1992):10832-10826) and radiation-induced E-selectin (WO 96/25947). However, said specific interaction with and uptake by the intended target cells is extremely difficult to achieve, for two reasons; i.e. (1) most of the proposed target molecules are also expressed on normal tissue, albeit at lower levels, and (2) it is difficult to construct targeted gene delivery vehicles. Many years of research have been invested by many different investigators in devising targeted gene delivery vehicles for this purpose, without significant success. Perhaps eventually this goal will be reached, but not without a major research effort and significant investment.
As a further alternative way to accomplish functional expression of genetic material in the vasculature of tumors, it has been proposed to transfer said genetic material into cultured endothelial cells ex vivo, followed by administration of said cultured endothelial cells via the circulation (WO 93/13807). This should result in selective incorporation of said cultured endothelial cells at sites of active angiogenesis, including the vasculature of solid tumors. However, such a selective incorporation into the vasculature of solid tumors has not been shown to occur.
Furthermore, the disadvantage of this approach is that it involves the isolation, ex vivo manipulation, and readministration of endothelial cells.
The present invention provides an effective and safe treatment of (solid) tumors in the body of mammals. This is accomplished by administration via the circulation of recombinant adenoviral vectors with wild-type infection spectrum that carry an interleukin-3 gene in a functional format.
Thus the invention provides the use of a recombinant adenoviral vector encoding IL-3 activity for manufacturing a pharmaceutical composition for the systemic treatment of tumors. For the present invention IL-3 activity is defined as the protein itself, derivatives and/or fragments thereof having at least, but preferably most or all of the biological functions of IL-3, although the amounts of activity displayed by these derivatives and/or fragments may vary. It is preferred that the systemic treatment is restricted to certain tissues, organs, or extremities, or certain combinations thereof, because adenovirus is in principle capable of infecting almost any cells in the host, so that the restriction enables to avoid unnecessary infection, as well as higher probability of infection of the proper targets. Thus in a preferred embodiment the invention provides a systemic treatment which includes isolated tissue perfusion. Tissue perfusion is intended to read on isolated tissues as well as organs and/or extremities or any combination thereof. Two approaches of isolated perfusion are provided, one whereby the isolated perfunded tissue includes the tumor and one whereby the isolated perfunded tissue excludes the tumor. In the second case organs or body parts which are liable to be damaged by the treatment or which are likely to influence the uptake of virus by the target cells can be excluded from the system to which the adenoviral vector encoding IL-3 activity is provided. A preferred organ to be excluded according to the invention is the liver.
In the other isolated perfusion route the vector is delivered to the isolated part only. It is preferred to deliver the vector in the form of a virus-like particle.
This means that the vector is packed in an adenovirus shell. The most preferred virus-like particle is the human homolog of recombinant adenovirus IG.Ad.CMV.rIL-3 deposited at the ECACC under accession number V96071634 or a functional derivative thereof.
IL-3 is not only capable of inducing regression of tumors, but it is also capable of retarding or halting the growth of tumors over prolonged periods of time. Many cytostaic agents are also capable of accomplishing regression of tumors, but are not capable of holding the regressed tumor in check over a prolonged period of time. It is therefor advantageous to make combinations of IL-3 activity and other cytostatic activity to have the best of both worlds. Regression of the tumor by administration of one or a number of doses of a cytotoxic agent and obtaining further regression as well as retarding or halting the growth of the regressed tumor by providing IL-3 activity.
Thus the invention further provides a means for treating tumors comprising a pharmaceutical composition comprising I1-3 activity and a pharmaceutical composition comprising cytostatic activity. Preferably, the IL-3 activity is provided by a recombinant adenoviral vector, (preferably in a virus-like particle) encoding said activity to be given systemically, either in an isolated perfusion format or not. Preferably the pharmaceutical composition comprising cytostatic activity is in a single dosage unit for injection into a solid tumor, to be given once or several times until the required dosage is reached.
Typically the virus-like particle is present in an amount of from about 106 to 5.109 iu in a perfusion fluid.
It is of course also possible that both activities are present in one composition.
Preferably the cytostatic or cytotoxic activity is TNF-activity, Melphalan, or adriamycin. The invention further provides a pharmaceutical composition for systemic treatment of tumors comprising IL-3 activity provided by a recombinant adenoviral vector encoding such activity, whereby said pharmaceutical composition is a perfusion fluid. Preferably the recombinant adenoviral vector is provided in the form of virus-like particles. Preferably said virus-like particles are present in an amount of about 106 to 5109 iu.
The most preferred virus is the human homolog of recombinant adenovirus IG.Ad.CMV.rIL-3 deposited at the ECACC under accession number V96071634 or a functional derivative thereof.
The invention further provides a kit of parts for the treatment of tumors comprising a pharmaceutical composition comprising IL-3 activity, means for isolating certain tissues, and means for perfunding said isolated tissues. Hereby the essential elements for performing a method of treatment according to the invention are given. The means for perfunding are preferably heart-lung machines or other equipment capable of perfunding and preferably oxygenating. Means for excluding certain organs, limbs and/or tissues are known in the art and references thereto can be found herein. If it is possible to exclude then it is of course also possible to limit perfusion to said organs, tissues or limbs which can be excluded. Of course the IL-3 activity in the kit of parts is again preferably provided by a recombinant adenoviral vector encoding said activity, preferably in the form of a virus-like particle, preferably present in an amount of about 106 to 5.109 iu. Preferably the kit of parts further comprises a pharmaceutical composition comprising cytostatic activity for the reasons already disclosed herein.
Despite the high potential of cancer gene therapy, the results of experimental treatment of solid tumors have until now been very disappointing. Direct injection of gene delivery vectors, mostly adenoviral vectors, carrying therapeutic genetic information into solid tumors has resulted in efficient gene transfer into tumor cells and has shown some, although still incomplete, tumor regression (e.g., see patent applications WO 95/05835 and EP 0 707071). The major limitation of this approach, however, has been that every solid tumor has to be individually injected. This makes clinical application of such a treatment far from realistic for most cancers, in particular for advanced cancers with metastases. The alternative approach, i.e. therapeutic gene delivery via the circulation after systemic intravascular administration of said gene delivery vector, has been associated with extremely low gene transfer efficiency into the tumor. The common belief in the field has been, therefore, that the gene transfer efficiency should be increased to obtain a significant therapeutic effect. It is also generally accepted that this should not be done by administering more gene delivery vectors, but by promoting the specific uptake of said gene delivery vector into the tumor cells or into the endothelial cells aligning the tumor vasculature. The reason for this is that high concentrations of untargeted gene delivery vectors cause (1) a stronger immune response, and (2) more toxicity due to delivery of the anti-tumor gene to other tissues. Furthermore, it is difficult and expensive to produce extremely high concentrations of gene delivery vectors.
The present inventors have made the surprising observation that adenoviral vector mediated delivery of an interleukin-3 gene through administration via the circulation into the vasculature of solid tumors results in a very effective cancer treatment. Said circulation is meant to include both the blood circulation and the lymphatic circulation. Said adenoviral vector is not treated in any way to promote its specific uptake by the solid tumor cells or the endothelial cells aligning the tumor vasculature. The therapeutic effect of said delivery is much more dramatic than could be expected from the low transduction efficiency (less than 1% transduced cells) that is obtained with said administration via the circulation. Established solid tumors growing in relevant animal models regressed completely. Said therapeutic effect is shown to be dependent on both said administration via the circulation and the biological activity of the interleukin-3 encoded by the introduced gene.
The present invention among other things provides a recombinant adenoviral vector that carries a nucleic acid molecule that encodes interleukin-3 or a functional derivative or a fragment thereof. Said nucleic acid molecule is provided with a format that allows functional expression of said interleukin-3 in solid tumor cells and/or in endothelial cells of the vasculature of a solid tumor in the body of a mammal after administration of said recombinant adenoviral vector to the circulation of said mammal. The term xe2x80x9cfunctional expressionxe2x80x9d is understood to mean production of said interleukin-3 with biological activity that leads to killing of said solid tumor cells. Said format is conferred upon said nucleic acid molecule by including upstream of said nucleic acid molecule an activator (promoter and/or enhancer) nucleic acid molecule that preferably interacts with one or more trans-activating transcription factors that are present in tumor cells or in cells of the vasculature of a tumor and downstream of said nucleic acid molecule a eukaryotic polyadenylation signal. Said activator molecule may be derived from the adenovirus used to construct said adenoviral vector or from a different adenovirus. Alternatively, said activator molecule is of exogenous origin. Useful activator molecules in this aspect of the invention are derived from, e.g. the Cytomegalovirus Immediate Early promoter/enhancer, the Rous Sarcoma Virus LTR promoter/enhancer, but may also be derived from other activator molecules known in the art. In this aspect of the invention it is preferred that said nucleic acid molecule encoding interleukin-3 is a functional derivative from or includes at least a functional fragment of a nucleic acid molecule isolated from the same species as said mammal. Because in most applications of the invention said mammal is a human, it is in most applications of the invention preferred that said nucleic acid molecule is a functional derivative from or includes at least a functional fragment of a nucleic acid molecule isolated from a human. The terms xe2x80x9cfunctional derivativexe2x80x9d and xe2x80x9cfunctional fragmentxe2x80x9d are used here to indicate that said nucleic acid molecule encodes a peptide molecule with the same biological activity in kind, but not necessarily in amount, as said interleukin-3. Many different examples of nucleic acid molecules encoding mutants of human interleukin-3 with functional interleukin-3 activity are given in European patent EP 0 413 383. It is furthermore preferred that the biological activity of said interleukin-3 includes the elicitation of an intense local inflammation associated with an inflammatory type cell infiltration. The recombinant adenoviral vectors according to the invention may be derived from any wild-type adenovirus serotype that allows the functional expression of said interleukin-3 in solid tumor cells and/or in endothelial cells of the vasculature of a solid tumor in the body of a mammal after administration of said recombinant adenoviral vector to the circulation of said mammal. In the examples given infra to illustrate the present invention said recombinant adenoviral vectors are derived from human adenovirus type 5. It is to be understood, however, that those skilled in the art will be able to apply other recombinant adenoviral vectors without departing from the invention. Methods for the construction of recombinant adenoviral vectors according to the invention and for their propagation on useful packaging cells have been described in patent applications EP 0 707 071 and WO 97/00326, incorporated herein by reference. Other examples of vectors and packaging systems useful in the invention include, but are not limited to, those given in patent applications WO 93/19191, WO 94/28152, WO 96/10642, and WO 97/04119.
The present invention furthermore provides a pharmaceutical composition that comprises the recombinant adenoviral vector defined supra in combination with a diluent that is not toxic to the recipient mammal at the dosage used and that retains sufficient stability of the infectivity of said recombinant adenoviral vector for a time long enough to allow uptake of said recombinant adenoviral vector into the solid tumor cells and/or endothelial cells of the vasculature of a solid tumor after administration of said composition to the circulation of the recipient mammal.
A typical non-limiting example of a diluent according to this aspect of the invention is an isotonic saline solution that is sterile and that is buffered at a physiological pH. Preferably, said diluent furthermore contains serum-substituting ingredients. In the examples given infra to illustrate the present invention Haemaccel (Behring Pharma) is used as a suitable diluent. It is to be understood, however, that those skilled in the art will be able to apply other diluents without departing from the invention. For some applications of the invention it is furthermore preferred that said pharmaceutical composition is oxygenated prior to administration. Optionally, said recombinant adenoviral vector (or virus) is prepared in lyophilized form. In the latter case, said recombinant adenoviral vector is suspended in solution to obtain said pharmaceutical composition before administering said pharmaceutical composition to the circulation of the recipient mammal. Typically, a pharmaceutical composition comprising one dose contains at least about 106, preferably about 108 infectious units (iu) of the adenoviral vector of the invention, but in certain conditions it is preferred that it contains at least about 109, more preferred 1010, or even more preferred 1011 iu. The amount of virus to be provided depends on many parameters. As disclosed herein only a very limited portion of the administered virus actually infects the target cells. This may be one reason to increase the amount of virus to be administered. Also the size of the tumor and/or the degree of its vascularization will influence the amount of virus required to get an effect. Another important aspect is of course the amount of IL-3 activity expressed by a cell infected with one or more viruses. This of course depends on the cell, but also on the promoter that drives the expression and its interaction with cell components of the expression machinery, etc.
Based on the rat studies, where a CMV promoter is driving the rat IL-3 gene, anti-tumor activity was measured after perfusion with 109 i.u. of IG.Ad.CMV.rIL-3. Perfusion time was 15 minutes. The size of this tumor was approximately 1 cm3 . Dose finding studies are performed, where the range of administered [perfused] will increase from 106 up to 1010 iu. Anti-tumor activity is measured according to the methods described. The lowest dose resulting in a maximal anti-tumor effect will be used to calculate the dose to be delivered to human tumors, assuming that the same promoter are used in the adenoviral vector harboring the human IL-3 gene or a derivative thereof. It is assumed that the infection of human cells by recombinant adenoviral vectors is 10xc3x97 more efficient than rat cells. Furthermore, we assume the vascular bed or the tumor to be proportional to the tumor volume. Therefore, the optimal dose assessed in the rat model is extrapolated to the human situation by the following calculation:       dose    ⁢          xe2x80x83        ⁢    delivered    ⁢          xe2x80x83        ⁢    to    ⁢          xe2x80x83        ⁢    humans    =                    [                  effective          ⁢                      xe2x80x83                    ⁢          dose          ⁢                      xe2x80x83                    ⁢          in          ⁢                      xe2x80x83                    ⁢          rat                ]            xc3x97              [                  tumor          ⁢                      xe2x80x83                    ⁢          volume          ⁢                      xe2x80x83                    ⁢                      (            human            )                          ]                    [              10        xc3x97        tumor        ⁢                  xe2x80x83                ⁢        volume        ⁢                  xe2x80x83                ⁢                  (          rat          )                    ]      
In another aspect, the invention provides a method to deliver said nucleic acid molecule that encodes interleukin-3 to solid tumor cells and/or endothelial cells of the vasculature of a solid tumor in the body of a mammal, whereby the adenoviral vector or pharmaceutical composition defined supra is administered to a site in the circulation of said mammal. Said circulation is meant to include both the blood circulation and the lymphatic circulation. Thus, the administration is performed to any site in the body of the recipient mammal where the blood or lymph fluids of said mammal pass. Preferred sites of administration are intravenous or intra-arterial, where it is further preferred that said administration is into an artery located upstream of the tumor vasculature. There are several means to perform said administration to the circulation. One of said means is by injection using, e.g., a syringe, a catheter or another infusion system known in the art. Preferably, said injection is performed at a controlled infusion rate. A much preferred means to perform said administration to the circulation is by perfusion. Perfusion is a technique whereby said administered pharmaceutical composition is caused to pass through said circulation or through a part of said circulation. When the administration is performed by perfusion it is furthermore preferred that said perfusion is done multiple times by creating a closed circuit and repassaging said pharmaceutical composition through said circulation or said part of circulation. Typically, said causing to pass is done by using a pump device and perfusion is performed at a rate depending on the species of the mammal to which said pharmaceutical composition is being administered. For humans, said rate is often in the range of approximately 40-80 ml/min and said perfusion is continued for a period of 60-90 minutes, but depending on patient, type of tumor, location thereof, these parameters may vary. For short treatment times (approximately 5-30 minutes) with the adenoviral construct an anoxic perfusion can be performed by those skilled in the art by using balloon catheters to make a closed circuit. No heart-lung machine is necessary.
In this aspect of the invention, said part of the circulation comprises the vasculature of the tumor or tumors to which gene delivery is performed. For optimal delivery of said nucleic acid molecule that encodes interleukin-3 to solid tumor cells and/or endothelial cells of the vasculature of a solid tumor it is preferred that the adenoviral vector or composition of the invention does not pass through the liver or a part of the liver of the recipient mammal. Thus, said part of the circulation does preferably not include the circulation of the liver or of a part of the liver, except when the tumor is located in or very close to the liver. For optimal delivery of said nucleic acid molecule that encodes interleukin-3 to solid tumor cells and/or endothelial cells of the vasculature of a solid tumor it is furthermore preferred that the blood of the mammal is first washed away from said closed circuit (e.g., by precirculation with the diluent of the pharmaceutical composition only) before said pharmaceutical composition is administered. Optionally, the blood that is washed away is collected and readministered at the end of the procedure. Surgical techniques for perfusion of parts of the circulation according to the present invention are under development and are already available for various specific parts of the circulation, such as, e.g., the liver (Fraker, D L et al., Circulatory shock, 44, p.45-50, 1994), the lung (Progrebniak H W et al., Ann. Thorac. Surg.,57, p.1477-83, 1994), and the kidney (Veen van de AH et al., Eur. J. Surg. Oncol. 20, p.404-405, 1994). A typical nonlimiting example of a routine perfusion technique useful in the invention is isolated limb perfusion (ILP), where a closed circuit is created between the femoral artery and the femoral vein. Alternatively, essentially the same perfusion techniques can be employed in the invention to exclude the delivery of said nucleic acid molecule to a part or parts of the circulation. In this aspect of the invention, the part or parts of the circulation to which said delivery is unwanted are perfused with a diluent according to the invention while said pharmaceutical composition is administered to the circulation systemically (hence, outside the perfusion circulation). An important example of this embodiment of the invention is exclusion of the liver circulation from delivery of said nucleic acid molecule.
The invention furthermore provides genetically modified solid tumor cells and cells of the vasculature of a solid tumor expressing said interleukin-3 in the body of a mammal. These cells expressing said interleukin-3 are obtained by administering the composition containing the adenoviral vector according to the invention using the method according to the invention via the circulation of said mammal.
The expression of said interleukin-3 in said solid tumor cells or cells of the vasculature of a solid tumor results in an effective killing of said cells. Thus, the present invention also provides a gene therapy treatment for solid tumors. All tumors that are in close contact with the circulation can be treated according to the invention. Although leukemias and lymphomas are not excluded, vascularized solid tumors are especially suited for treatment according to the invention. Examples of types of said solid tumors include, but are not limited to, carcinomas (e.g., of the lung, bladder, kidney, breast, stomach, pancreas, urogenital tract, and intestine), sarcomas (e.g., soft tissue sarcomas, osteogenic sarcomas, or Kaposi""s sarcoma), gliomas and melanomas. Also benign types of tumors, such as, e.g., angiomas and fibrocytomas can be treated according to the invention. It is to be understood, however, that the scope of the present invention is not to be limited to the treatment of any particular type of tumor.
It is furthermore to be understood that the cancer treatment according to the invention may be combined with other methods of cancer treatment known in the art. Such treatment combinations are also part of the present invention.
The invention is illustrated by means of the following examples. It is to be understood that said examples are not meant to limit the scope of the invention in any way.
Example 1 teaches the production of adenoviral vectors and pharmaceutical compositions according to the invention.
Example 2 teaches the cloning and production of an adenoviral vector with the human IL-3 gene and the pharmaceutical composition according to the invention.
Example 3 and 4 show the gene transfer efficiency that is obtained when adenoviral vectors are administered to a solid tumor via the circulation or by direct intra-tumor injection, as well as the unwanted gene transfer into non-tumor cells in both cases, and the type of cells in the tumor that are transduced using these administration methods. It is shown that the direct injection results in approximately 87-times more expression of the introduced gene in the tumor than administration via the circulation. The direct injection efficiently transduces many tumor cells along the needle tract, whereas administration via the circulation mainly transduces endothelial cells of the tumor vasculature, a few solid tumor cells adjacent to the vascular endothelial cells and some cells in or near the capsule of the tumor. Gene transfer into tissues other than the tumor hardly occurs using either method.
Examples 5, 6, 7 and 8 clearly demonstrate the effective anti-tumor effect that is accomplished by administering an adenoviral vector carrying the interleukin-3 gene into two types of solid tumors via the circulation. Complete regression of said tumors occur. Experiments show that this effective anti-tumor effect is not obtained by direct intra-tumor injection or by using an adenoviral vector that expresses the IL-3 gene at low levels. Control isolated limb perfusion experiments show that this effective anti-tumor effect is not obtained by a isolated limb perfusion without addition of said adenoviral vector with the interleukin-3 gene or by treatment with an adenoviral vector without effector gene. After the latter treatment some delay in tumor growth is observed when ROS-1 osteosarcomas were used, but the tumors do not regress. The latter growth delay is not observed when BN175 tumors were treated. Example 5 shows that the anti-tumor effect is specific for the activity of the interleukin-3 gene. Anti-cancer treatment by administering adenoviral vectors expressing the HSV-tk gene via the circulation followed by ganciclovir injections show only incomplete effects.
Example 9, 10 and 12 demonstrate that in the rats with the two tumor models studied the optimal dose for administering of the adenoviral vector carrying the interleukin-3 gene via the circulation is 1.09 iu (infectious units). And that a 15 minutes perfusion results in good antitumor effects.
Example 11 clearly demonstrates that the administration via the circulation of 1.109 iu of the adenoviral vector carrying the interleukin-3 gene is at least as efficient as the established combination therapy with TNF( and Melphalan).